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Looking through the ice: Quality control of cryopreserved cells and tissues with cryo focus ion beam scanning electron microscopy

 
: Katsen-Globa, A.; Meiser, I.; Putz, D.; Cismak, A.; Heilmann, A.; Zimmermann, H.

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Cryobiology 61 (2010), Nr.3, S.393-394
ISSN: 0011-2240
ISSN: 1090-2392
Englisch
Zeitschriftenaufsatz
Fraunhofer IBMT ()

Abstract
Modern cryobanking for long-term storage of large numbers of objects in or over liquid nitrogen needs regular and exact quality control without thawing the materials. Cryo Focus Ion Beam combined with Cryo Scanning Electron Microscopy (Cryo-FIB-SEM) is useful. This method allows examination of cell surface and, with FIB-preparation, three-dimensional analysis of the intracellular ultra-structure. Further, it permits estimation of adhesion contacts of the cells with frozen cryosubstrates. Cryo-FIB-SEM investigation can be performed in a closed cryotechnological chain during: (a) controlled freezing of materials; (b) storage at low temperatures; (c) sample transfer to Cryo-FIB-SEM; and (d) return of objects to the cryobank. Some difficulties have to be overcome. Firstly, additional ice formation during sample transfer to the Cryo-FIB-SEM examination has to be prevented. Secondly, the temperature of the sample holder must be constant during examination. Thirdly, the biological objects have to be contrasted to distinguish them from ice.
RIN-m multi-cellular spheroids producing insulin or RIN-m cell monolayers grown on plastic substrates were treated with electron contrast agents without any loss of vitality. Afterwards the samples were mounted on specially constructed object holders and frozen with cryoprotectants in a computer-controlled freezer at 1 °C/min from 4 °C to ?80 °C. The sample transfer to Cryo-FIB-SEM was carried out in a conventional cryo-dry-shipper (?160 °C) with a static holding time of 12 days. From the dry-shipper to the SEM cryo-chamber the samples were transported in newly developed Teflon wrapping.
We have demonstrated differences between various cryopreservation methods. Numerous extracellular ice crystals could be observed in samples preserved by slow freezing procedures while Cryo-FIB-SEM of vitrified biomaterials confirms absence of ice. Usage of contrast agents has improved cryopreservation of spheroids most likely due to plasma membrane enhancement but no differences could be recognized between control and cryopreserved samples. By studying contrasted and frozen RIN-m monolayer fine chromatin structures of mitotic cells could be seen through the ice. The results can be considered as a first step in the development of a new class of cryo-imaging techniques.

: http://publica.fraunhofer.de/dokumente/N-150070.html